Journal: The Journal of Cell Biology

Article Title: SLX4–XPF mediates DNA damage responses to replication stress induced by DNA–protein interactions

doi: 10.1083/jcb.202003148

Figure Lengend Snippet: Various DDR proteins are recruited to the LacI-bound lacO array in an S-phase–specific manner. (A) A schematic is shown of the rapid and controlled induction of LacI binding to lacO in U2OS 40–2-6 cells stably expressing HA-ER T2 -LacI (ΔNLS). Nuclear accumulation of HA-ER T2 -LacI (ΔNLS) was stimulated by treatment with 4-OHT. Representative images are shown of U2OS 40–2-6 ER-LacI cells treated with 4-OHT or vehicle only (EtOH) for 30 min. LacI was detected with anti-LacI immunostaining (red) and DNA with DAPI staining (blue). White arrows indicate LacI foci. Scale bar, 10 µm. (B) U2OS 40–2-6 ER-LacI cells were treated with 1 µM 4-OHT or vehicle-only (EtOH) for 80 min and labeled with 10 µM BrdU for the last 20 min. BrdU incorporation was then examined by DNA immunoprecipitation with anti-BrdU antibody (BrdU-IP), followed by quantitative PCR analysis using primer pairs to detect the lacO sequences or LMNB2 replication origin. The relative incorporation of BrdU into the lacO sequences compared with the control region ( LMNB2 origin) was calculated. The means ± SD are shown ( n = 6). *, P < 0.05 (two-tailed Student’s t test). (C and D) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT for 2 h were double immunostained with the indicated antibodies and counterstained with DAPI. ssDNA was detected with anti-BrdU antibody under nondenaturing conditions. (C) Representative images are shown. Yellow and white arrows indicate colocalization and noncolocalization of DDR proteins with the LacI foci, respectively. Scale bar, 10 µm. (D) Colocalization frequencies of the indicated foci with the LacI foci are shown. Values were calculated from the sum scores of at least two independent experiments. (E) U2OS 40–2-6 ER-LacI cells were synchronized in G1 phase by lovastatin treatment or in S phase by HU treatment with subsequent release for 4 h (for details, see Materials and methods), then treated with 1 µM 4-OHT for 2 h and double immunostained, followed by DAPI staining and analysis. Colocalization frequencies were calculated from the sum scores of two independent experiments. ***, P < 0.001 (χ 2 test). Individual data points from the two experiments are also depicted.

Article Snippet: The following drugs were used: 4-OHT (Abcam), RO-3306 (Sigma), HU (Sigma), lovastatin (LKT Laboratories), VE-821 (Haoyuan Chemexpress), KU-55933 (Sigma), and mirin (Sigma).

Techniques: Binding Assay, Stable Transfection, Expressing, Immunostaining, Staining, Labeling, BrdU Incorporation Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction, Control, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: SLX4–XPF mediates DNA damage responses to replication stress induced by DNA–protein interactions

doi: 10.1083/jcb.202003148

Figure Lengend Snippet: Cell-cycle synchronization of U2OS 40–2-6 ER-LacI cells and the effect of synchronization on the DDRs induced by LacI. (A and B) G1-phase synchronization. U2OS 40–2-6 ER-LacI cells were treated with 40 µM lovastatin for 40 h and then subjected to FACS (A) and SDS-PAGE followed by immunoblotting (B). Coomassie Brilliant Blue staining served as the loading control. G1 synchronization was confirmed by the reduction in cyclin A and cyclin E protein levels (B). Data are representative of two independent experiments. (C) S-phase synchronization. U2OS 40–2-6 ER-LacI cells were synchronized with 2.5 mM HU for 18 h and released into fresh medium. Cell-cycle distribution was analyzed by flow cytometry at the indicated times after release. Data are representative of two independent experiments. (D) Asynchronous cells or G1-synchronized cells as described in (A) were treated with 1 µM 4-OH-tamoxifen (4-OHT) for 4 h or 6 h. Colocalization frequencies of the indicated foci with LacI foci were analyzed as described in . (E) U2OS 40–2-6 ER-LacI cells were synchronized in G1 phase or in S phase as described in , treated with 1 µM 4-OHT for 2 h, and double immunostained, followed by DAPI staining. Colocalization frequencies of the indicated foci were calculated from the sum scores of two independent experiments. n.s., not significant (χ 2 test). Individual data points from the two independent experiments are also presented. (F–H) G0-phase synchronization. Cells synchronized at G0 phase by serum starvation (0.1% FCS) for 72 h were subjected to FACS analysis (F) and immunoblotting for cyclin A (G). In G0 cells, cyclin A levels were reduced. After 72 h serum starvation, cells were treated with 1 µM 4-OHT for 6 h, and the colocalization frequencies of the indicated DDR factors with LacI were analyzed (H). Compared with the values with asynchronous cells (e.g., those shown in D), the accumulation frequencies were lower. (I) Cells arrested in early S phase as described in C were treated with 1 µM 4-OHT and either left arrested with HU or released into fresh medium for 6 h. Colocalization frequencies were calculated from the sum scores of two independent experiments. ***, P < 0.001; n.s., not significant (χ 2 test). Individual data points from the two independent experiments are also shown. CBB, Coomassie Brilliant Blue.

Article Snippet: The following drugs were used: 4-OHT (Abcam), RO-3306 (Sigma), HU (Sigma), lovastatin (LKT Laboratories), VE-821 (Haoyuan Chemexpress), KU-55933 (Sigma), and mirin (Sigma).

Techniques: SDS Page, Western Blot, Staining, Control, Flow Cytometry

Journal: bioRxiv

Article Title: Rewiring glucose metabolism improves 5-FU efficacy in glycolytic p53-deficient colorectal tumors

doi: 10.1101/2021.11.11.468185

Figure Lengend Snippet: A) Representative brightfield images of WT and CRC organoids treated with 5-FU for 7 days (scale bar = 500 μm). B) Cell viability analysis of WT and CRC tumor organoids by flow cytometry to distinguish alive (DAPI - ) from death cells (DAPI + ) upon 7 days of 5-FU treatment (mean ± SEM, n = 3-5, ANOVA, Sidak’s multiple comparisons test). C) Western blot detection of γH2AX and vinculin in lysates from WT and CRC tumor organoids treated with 5-FU for 48 hours (representative for n = 5). D) Quantification of cells with DNA damage by flow cytometry of WT and CRC organoids treated with 5-FU for 48 hours and stained with anti-γH2AX (mean ± SEM, n = 3-8, ANOVA, Sidak’s multiple comparisons test). E,F) , Western blot detection of (p)Chk1, (p)Chk2, γH2AX and vinculin of lysates from WT and APKS organoids treated with 5-FU for 24 hours and co-treated with either ATR inhibitor VE-821 or ATM inhibitor KU55933 or both for 26 hours (representative for n = 3, WT: Chk2: Santa Cruz, #SC-9064, APKS: Chk2: Cell Signaling, #3440). ns: non-significant, * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Treatments with ATR inhibitor VE-821 (5 μM, Bioconnect, #S8007) and ATM inhibitor KU-55933 (10 μM, Sigma, #SML1109) were started 2 hours before 5-FU administration.

Techniques: Flow Cytometry, Western Blot, Staining